SUMMARY Hepatitis B Virus (HBV) is a major cause of liver disease. Assembly of the HBV capsid is a critical step in virus production and an attractive target for new antiviral therapies. We determined the structure of HBV capsid in complex with AT-130, a member of the phenylpropenamide family of assembly effectors. AT-130 causes tertiary and quaternary structural changes, but does not disrupt capsid structure. AT-130 binds a hydrophobic pocket that also accommodates the previously characterized HAP compounds, but favors a unique quasi-equivalent location on the capsid surface. Thus, this pocket is a promiscuous drug binding site and a likely target for different assembly effectors with a broad range of mechanisms of activity. That AT-130 successfully decreases virus production by increasing capsid assembly rate without disrupting capsid structure delineates a new paradigm in antiviral design, that disrupting reaction timing is a viable strategy for assembly effectors of HBV and other viruses.

Understanding the biological self-assembly process of virus capsids is key to understanding the viral life cycle, as well as serving as a platform for the design of assembly-based antiviral drugs. Here we identify and characterize the phenylpropenamide family of small molecules, known to have antiviral activity in vivo, as assembly effectors of the Hepatitis B Virus (HBV) capsid. We have found two representative phenylpropenamides to be assembly accelerators, increasing the rate of assembly with only modest increases in the stability of the HBV capsids; these data provide a physical-chemical basis for their antiviral activity. Unlike previously described HBV assembly effectors, the phenylpropenamides do not misdirect assembly; rather, the accelerated reactions proceed on-path to produce morphologically normal capsids. However, capsid assembly in the presence of phenylpropenamides is characterized by kinetic trapping of assembly intermediates. These traps resolve under conditions close to physiological, but under conditions which favor phenylpropenamide binding and strong core protein-protein interactions, we found that trapped intermediates persist. The phenylpropenamides serve as chemical probes of the HBV capsid assembly pathway by trapping on-path assembly intermediates, illustrating the governing influence of reaction kinetics on capsid assembly.

Though the hepatitis B virus (HBV) core protein is an important participant in many aspects of the viral life cycle, its best-characterized activity is self-assembly into 240-monomer capsids. Small molecules that target core protein (core protein allosteric modulators [CpAMs]) represent a promising antiviral strategy. To better understand the structural basis of the CpAM mechanism, we determined the crystal structure of the HBV capsid in complex with HAP18. HAP18 accelerates assembly, increases protein-protein association more than 100-fold, and induces assembly of nonicosahedral macrostructures. In a preformed capsid, HAP18 is found at quasiequivalent subunit-subunit interfaces. In a detailed comparison to the two other extant CpAM structures, we find that the HAP18-capsid structure presents a paradox. Whereas the two other structures expanded the capsid diameter by up to 10 Å, HAP18 caused only minor changes in quaternary structure and actually decreased the capsid diameter by ϳ3 Å. These results indicate that CpAMs do not have a single allosteric effect on capsid structure. We suggest that HBV capsids present an ensemble of states that can be trapped by CpAMs, indicating a more complex basis for antiviral drug design. IMPORTANCEHepatitis B virus core protein has multiple roles in the viral life cycle-assembly, compartment for reverse transcription, intracellular trafficking, and nuclear functions-making it an attractive antiviral target. Core protein allosteric modulators (CpAMs) are an experimental class of antivirals that bind core protein. The most recognized CpAM activity is that they accelerate core protein assembly and strengthen interactions between subunits. In this study, we observe that the CpAM-binding pocket has multiple conformations. We compare structures of capsids cocrystallized with different CpAMs and find that they also affect quaternary structure in different ways. These results suggest that the capsid "breathes" and is trapped in different states by the drug and crystallization. Understanding that the capsid is a moving target will aid drug design and improve our understanding of HBV interaction with its environment. Hepatitis B virus (HBV) causes degenerative liver disease and is the leading cause of liver cancer, with 240 million chronically infected individuals (1, 2). Current antiviral therapies control the progression of the disease but fail to eliminate the virus (3, 4). There is a need for improved therapies to combat chronic infections. One approach is to target virus assembly (5-7).HBV is an enveloped, double-stranded DNA virus with an icosahedral nucleoprotein core. The HBV capsid is the protein shell of the core. Beyond genome protection, the capsid is involved in intracellular trafficking, interaction with nuclear import machinery, regulation of reverse transcription, signaling, completion of reverse transcription, RNA chaperoning, and envelope acquisition (8). Capsid assembly is central to HBV replication. In vivo HBV capsids assemble around an RNA copy of the viral g...

bAssembly effectors are small molecules that induce inappropriate virus capsid assembly to antiviral effect. To identify attributes of hepatitis B virus (HBV) assembly effectors, assembly reaction products (normal capsid, noncapsid polymer, intermediates, and free dimeric core protein) were quantified in the presence of three experimental effectors: HAP12, HAP13, and AT-130. Effectors bound stoichiometrically to capsid protein polymers, but not free protein. Thermodynamic and kinetic effects, not aberrant assembly, correlate with maximal antiviral activity. Hepatitis B virus (HBV) is a global public health problem. According to World Health Organization estimates, 360 million people suffer from chronic HBV infection, contributing to approximately 600,000 deaths every year (1). HBV-specific antiviral drug development has focused on targeting the viral reverse transcriptase (RT). However, RT inhibitors do not usually clear HBV infection, even after prolonged treatment (2-4). Furthermore, cessation of RT inhibitors can lead to life-threatening viral flares; therefore, they are generally a lifelong therapy (5). An alternative therapeutic target is highly desirable. One attractive target is assembly of HBV's capsid from core protein.HBV is a DNA virus composed of a protein-studded lipid envelope surrounding an icosahedral nucleoprotein core (6, 7). The protein shell of the core, the capsid, is a Tϭ4 icosahedral complex built from 120 copies of core protein homodimer. The core protein is a 183-amino-acid protein comprised of a 149-residue assembly domain (which includes the dimerization motif) and a C-terminal 34-residue RNA binding domain which is not required for assembly (8). The core protein assembly domain has no human homolog (9). The assembly domain is referred to as Cp149. In the HBV life cycle, like many icosahedral viruses, the capsid has critical roles in virus replication, making it an excellent target for antiviral therapy (10, 11). Cp149 assembly is a function of protein concentration, ionic strength, and temperature (12). A molecule that modulates capsid assembly could interfere with the geometry of core protein interaction, packaging viral nucleic acid, and the stability of newly assembled virions (13-16). A number of HBV assembly effectors have been investigated (17-21). Recently, capsid assembly has also been targeted in other viral systems, including .Two classes of HBV assembly effectors have been discovered in searches for nonnucleoside inhibitors of HBV replication, the heteroaryldihydropyrimidines (HAPs) and phenylpropenamides (25)(26)(27)(28)(29). On the basis of observations with purified Cp149, HAPs increase the kinetics of assembly and strengthen dimer-dimer association to stabilize capsids, and at high concentrations, they misdirect assembly (14,20). On the basis of a crystal structure of the HAP-HBV complex, a series of HAPs with different properties were designed; their effects on the thermodynamics and kinetics of assembly of purified Cp149 were compared with inhibition of virion product...